Abstract Pluripotent stem cells (PSCs) are an important tool for modeling developmental disorders and a critical resource for regenerative medicine. The path between pluripotency and a differentiated state involves a series of binary fate choices, which are difficult to control ex vivo. With single-cell transcriptomics, it is clear at the time of these choices, cells show gene expression heterogeneity with co-expression of transcription factors typically associated with each of their alternative fates. This form of heterogeneity or noise in expression is a substrate for probabilistic cell-fate decisions; modulation of this noise can therefore bias the decision outcome. Mouse embryonic stem cells (mESCs) have been well- studied in this context with focus on the pluripotency factor NANOG, which can have a heterogeneous and dynamic expression pattern. There is a gap in knowledge as to how the expression noise of fate-determining genes, like Nanog, are tuned. The overall goal is to identify and perturb the mechanisms regulating noise of fate-determining genes to increase the efficiency of stem cell differentiation and iPSC generation. Gene expression variability partly arises from the episodic or bursty nature of transcription. The main objective of this proposal is to identify how mESCs tune transcriptional bursting, and thus noise, of the Nanog promoter. To this end, through a small-molecule screen, I have identified a compound capable of decreasing burst frequency and increasing burst size of the Nanog promoter, therefore enhancing NANOG protein noise without changing its mean level in mESCs. We have thus perturbed a mechanism regulating transcriptional bursting of the Nanog promoter and, in the process, amplified expression noise on both mRNA and protein levels. Elucidating this mechanism will provide insight into the determinants of transcriptional noise for a key, fate- determining gene and open new avenues for optimization of processes like stem cell differentiation or reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Previous data show that remodeling of promoter-occupying nucleosomes can be the rate-limiting step defining burst frequency; thus, increasing nucleosome occupancy of the promoter can decrease burst frequency and increase expression noise. Alternatively, it has been shown that the strength of looping between a promoter and its respective enhancers determines burst frequency. Taken together, I hypothesize that the transcriptional noise of Nanog is amplified either through increased nucleosome occupancy of the promoter or through rewiring of enhancer-promoter contacts. Aim 1 will characterize the changes in chromatin organization that occur within the NANOG locus during transcriptional noise amplification. Additionally, I hypothesize that NANOG-noise amplification in somatic cells can potentiate responsiveness to reprogramming cues. Aim 2 will clarify how noise enhancement of fate-determining genes can synergize with existing reprogramming protocols to enhance iPSC generation. Uncovering these noise-amplification mechanisms will reveal principles underlying transcriptional bursting and enable more efficient use of iPSCs as a tool for disease modeling, drug discovery and tissue regeneration.